High-frequency and strong FAD2 co-suppression：A bottleneck to increase polyunsaturated fatty acids in seeds.

Fatty acid desaturase2 (FAD2) catalyses the conversion of oleic acid to linoleic acid and is the main determinant of the levels of essential poly-unsaturated fatty acids (PUFAs) in seed oils. The very limited number of successful examples of overexpression of FAD2 over the last two decades and a shortage of reports on co-suppression make it uncertain whether FAD2 can increase PUFAs effectively across a broad range of oil crops. In this study, strong co-suppression was observed in about 80% of over 100 transgenic lines when FAD2 was overexpressed in three oilseed crops, namely flax (Linum usitatissimum), carinata (Brassica carinata), and camelina (Camelina sativa), as well as in the model plant Arabidopsis. Further analyses of Arabidopsis transgenic lines revealed both endogenous and transgenic FAD2 gene-silencing. Thus, the commonality and potency of FAD2 co-suppression seemingly imposes an obstacle to engineering oilseed PUFA enhancement by direct FAD2 overexpression. AtFAD2, driven by the 35S promoter, also caused co-suppression in Arabidopsis roots. The FAD2 co-suppression was unstable and PUFA phenotypes of T4 lines were similar to the wild-type, further indicating that high PUFA content cannot be achieved by screening advanced generations. However, we demonstrate that the obstacle of FAD2 co-suppression can be overcome in the Arabidopsis rdr6 mutant, which is impaired in post-transcriptional gene-silencing, and that lines with high PUFA content are stable through four generations.